Effect of gamma rays on the essential oil and biochemical characteristics of the Satureja mutica Fisch & C. A. Mey

There are 16 species in the genus Satureja L. (Lamiaceae), of which 10 are native. This research aimed to investigate the effect of gamma rays and storage conditions and duration on the percentage and components of the essential oil and some biochemical characteristics of Satureja mutica Fisch & C.A. Mey at the Research Institute of Forests and Rangelands. Plants were collected at the full flowering stage and exposed to different doses of gamma rays (0, 2.5, 5, 7.5, and 10 kGy) at the Atomic Energy Organization, Iran. The samples were kept in a refrigerator (4 °C) and in the shade (25 ± 2 °C) for 0, 120, and 240 h. This experiment was performed in a completely randomized design. Essential oil extraction was done by water distillation for 2 h. The composition of their essential oil components was identified using GC and GC/MS. Some biochemical traits, including phenol content, antioxidant capacity, and carbohydrate content, were measured. The results indicated that irradiation on the percentage of essential oil showed a statistically significant difference. In addition, the interaction effect of irradiation × storage conditions, irradiation × duration of storage, on the percentage of essential oil was significant. According to a comparison of the means, 2.5 kGy irradiation produced the highest percentage of essential oil (0.4%); in contrast, a significant decrease was detected in components with 7.5 and 10 kGy irradiation. It was observed that the percentage of some essential oil compounds decreased with the gamma-ray intensity increase. 2.5 kGy of gamma rays and shade storage conditions for 240 h led to the highest content of p-cymene and carvacrol. Nevertheless, the highest thymol content was obtained under refrigeration conditions without irradiation. The maximum phenol content and antioxidant capacity were obtained when the plants were irradiated with 2.5 and 7.5 kGy gamma rays. However, the maximum carbohydrate rate was observed in non-irradiated plants. It was concluded that low-intensity gamma rays could improve the percentage of essential oil and main components like p-cymene and carvacrol in S. mutica Fisch & C.A. Mey.

www.nature.com/scientificreports/ 10 m, an inner diameter of 0.1 mm, and a stationary phase layer thickness of 0.4 µ.The injection chamber temperature was set at 280 °C, and the detector temperature was set at 280 °C.The thermal programming of the column included raising the temperature from 60 to 285 °C with an increase rate of 40 °C/min and then keeping it at 285 °C for 3 min.Helium was used as a carrier gas at 0.5 mL/min.An Agilent 7890A gas chromatograph connected to an Agilent 5975C quadrupole mass spectrometer (USA), equipped with DB-5 column (length 30 m, inner diameter 0.25 mm, and stationary phase layer thickness equal to 0.25 µ) for qualitative analysis was used.The thermal programming of the column includes increasing the temperature from 60 to 220 °C with an increased rate of 3 °C/min and then increasing it to 260 °C with an increased rate of 20 °C/min, and finally, it is kept at this temperature for 3 min.The injection chamber was set at 260 °C and the transfer line at 280 °C.Helium was used as a carrier gas with a flow rate of 30.6 cm/s.The scanning time was one second, and the ionization energy was 70 electron volts and 40 to 340 m/z.The inhibition period, Quats inhibition index, mass spectra comparison with standard compounds, and employing device library information were used to identify constituents in essential oils 20 .

Biochemical parameters determination
Furthermore, several biochemical parameters were measured, including antioxidant activity, phenol, and carbohydrate content.

Phenol content
Folin-Ciocalteu's phenol reagent was used to measure total phenol content.The extract was prepared at a concentration of 10 ml/mg, and then 0.5 ml of each extract was mixed with 2.5 ml of 0.2 normal Folin-Ciocalteu's phenol reagent and stirred for 5 min.Then, a 2 ml sodium carbonate solution of 10% at 75 g/l was added.The absorbance of the samples was measured after leaving them at room temperature for 2 h with an ultraviolet spectrophotometer at a wavelength of 760 nm against a blank (methanol).The amounts of total phenol in the extract are measured using a standard curve based on mg gallic acid /g of extract 21 .

Carbohydrate content
Carbohydrates were extracted using the 95% ethanol and sulfuric acid method.For this purpose, 0.2 g fresh leaf tissue was heated with 10 cc of 95% ethanol for one hour in a Bain-Marie bath at 80 °C.One cc of 0.5% phenol and 5 cc of 98% sulfuric acid were added to each cc of this sample.The amount of absorbed light was measured at 483 nm using a spectrophotometer.The amount of extracted carbohydrates was obtained based on micrograms of glucose per gram of weight from the standard table.

DPPH free radical inhibition
Evaluation of leaf extract antioxidant activity was done by using DPPH method.For this purpose, first, 2.5 ml of methanolic solution of leaf extract was poured into a test tube.Then, 1 ml of methanolic DPPH solution was added to it.The contents of each tube were thoroughly vortexed.After 30 min, at room temperature and in a dark environment, their absorbance at 518 nm was read with a spectrophotometer against a blank containing methanol.In this method, vitamin C and BHT were used as positive controls.DPPH free radical inhibition percentage was calculated using the following equation: where A0 is the absorbance of the control and As, is the absorbance of the sample.The results were expressed as IC50 (an amount of antioxidant required for DPPH concentration to reach50% of the initial value) 22 .

Statistical analysis
The sources of variation include gamma radiation intensity at 4 levels: 0, 2.5, 7.5, and 10 kGy, storage conditions at 2 levels: room temperature of 25 °C and refrigerator at 4 °C; and storage duration factor at 3 levels: 0, 120 and 240 h.As a result, the number of studied treatments, including eight repetitions for each treatment, was equal to 92 experimental units, which were analyzed in a factorial format using a completely random design.The data in this study are presented as mean values + standard deviation (S.D.) based on three replications, analyzed, and compared using the LSD test at 0.05.

Ethical statement
This research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors.

Essential oil compositions
Analyzing the variance of the effects of gamma rays on essential oil composition and percentage showed that the simple effect of gamma irradiation was significant at the level of 5% on essential oil percentage.At the 5% level, the direct effect of irradiation on storage conditions and the interaction between irradiation and storage on the percentage of essential oil was significant.A significant effect was also observed based on the influence of irradiation, conditions, and duration of storage, especially on the main three compounds of p-cymene, thymol, and carvacrol.Additionally, it was shown that most essential oil components, particularly the three main compounds of p-cymene, thymol, and carvacrol, varied significantly according to irradiation, storage conditions, and duration (Table 1).
As shown in Fig. 1 although there was no statistically significant difference between the 2.5 kGy treatment and the control at 5 kGy, a comparison of the means revealed that irradiation with 2.5 kGy of gamma rays produced the highest percentage of essential oil.However, as the irradiation intensity increased, the essential oil amount decreased.
Figure 2 shows that there is no clear statistical trend.The treatment of 2.5 kGy in 240 h of storage had the highest percentage of essential oil, according to a comparison of the mean effects of various gamma dosages and storage periods (Fig. 3).
The amount of essential oil and the components of S. mutica essential oil were examined during the current study's examination of the interactions between various gamma radiation intensities, time, and storage circumstances.P-cymene, one of the important components of the essential oil and the precursor of carvacrol of S. mutica, had its highest amount when the plant was exposed to gamma rays at 2.5 kGy as well as its highest content in the shade and 240 h (more than 45%), so it seems that during the storage period as well as the treatment with low doses of gamma rays, the conversion of precursors to p-cymene occurs (Table 2).
It can be deduced that in the event of irradiation and keeping the plant under the conditions described above, we will see a decrease in the amount of thymol.This can be related to the exit of this substance from the plant.Satureja mutica essential oil also contains thymol, responding negatively to gamma rays.At every level of this radiation, the proportion of the essential oil ingredient was minimized.However, after the plant was exposed to 2.5 kGy of gamma rays and kept in the refrigerator for 120 h, the amount of carvacrol in S. mutica essential oil reached its greatest value (17.42%) (Table 2).

The impact of storage time and duration and gamma radiation exposure on the biochemical traits of S. mutica Fisch & C. A. Mey
The results of the analysis of variance of the effect of gamma rays, time, and duration of storage on the biochemical traits of S. mutica Fisch & C. A. Mey are given in Table 3.As can be seen, each of these has a considerable impact on biochemical traits such as phenol content, antioxidant capacity, and carbohydrate.Furthermore, it affects the interaction between gamma rays × storage time × duration.These findings showed that the phenol content rose over time, reaching its peak (16.76 µg/ml) after 240 h of storage in the shade with 2.5 kGy of gamma radiation.The amount of total phenol dropped as the gamma radiation dosage increased.As a result of a high radiation dosage, lower phenol content was extracted so that the least of it was observed at 10 kGy gamma radiation (10.33 µg/ml) in refrigerator storage after 240 h (Table 3).
Under conditions of refrigerator storage, 240 h of storage period, and 7.5 kGy of gamma radiation, the highest concentration of antioxidant capacity was discovered.After 240 h, 10 kGy treatment showed a significant difference in antioxidant capacity (77.5%) from the control, whereas 5 kGy treatments showed a marked decline in antioxidant capacity (51.38%) (Table 3).
Table 3 shows that the radiation-free condition had the highest carbohydrate content.Additionally, the findings indicated that carbohydrates decreased during storage time.Nonirradiated samples exhibited a higher total carbohydrate content (average values considering all irradiated materials (8.66 µg/ml extracted) than the corresponding irradiated samples).In particular, depending on the increased radiation dose, carbohydrate levels decreased significantly.Irradiated samples exhibited lower total carbohydrate content (average values considering 10 kGy irradiated materials in the refrigerator for 240 h = 3.4, 3.7, and 4.61 µg/ml extracted, respectively), while lower radiant (5 and 10 kGy) resulted in a significant increase in carbohydrate levels.

Discussion
Research results show that nuclear technologies significantly affect morphology and phytochemistry to boost agricultural production and improve quality.Several researchers have compared the active components of irradiated medicinal plants with control samples, but only a small number of those studies have used high intensities to detect changes in the principal constituents of the treated plants 23 .Gamma radiation is often called a cold process in which the processed material's temperature does not significantly increase.This method is, therefore, suitable for heat-sensitive items like medicinal plants.
Based on this study, the essential oil (EO) percentage obtained from the aerial parts of S. mutica Fisch & C. A. Mey with 2.5 kGy gamma radiation was higher.In addition, it was affected by 2.5 kGy gamma ray doses and storage duration after 240 h.
The present study describes the essential oil profile and biochemical changes in the main compounds of p-cymene, thymol, and carvacrol that emphasize the appropriate treatments and subjects' selection; it also shows that drying before hydrodistillation resulted in losses or increases of essential oil, so the issue has been further investigated.The subject and treatments were appropriately chosen when there were noticeable differences between treatments in the percentage of essential oil and the main compounds of p-cymene, thymol, and carvacrol 15 .
Vol.:(0123456789)In agree with our results, the content of bioactive components increased in Artemisia 24 , white mushrooms 25 , and Carum carvi L. 26 when exposed to gamma rays.Although, high intensities have a decreased yield in plants like barley 27 , but low doses have an increased yield.As a result, to increase plant performance, one should act after knowing the impact of gamma radiation on the intended product and its optimal intensity level.According to Haddad et al. 28 , low levels of gamma rays positively affect the percentage and composition of essential oils from medicinal plants like thyme, lavender, and eucalyptus.This research had similar results to this research.These outcomes are consistent with those reported by Tanacetum annuum L. 29 , Thymus vulgaris L. 30 ; Syzygium aromaticum and Cinnamomum zeylanicum 31 .
According to our finding, 16 essential oils were extracted from S. mutica Fisch and C. A. Mey after gamma radiation.Carvacrol and P-cymene make up the majority of the oil.The essential oil of S. mutica Fisch & C. A. Mey gathered from north and northwest Iran has 48 and 46 components, respectively 32 .Thymol was the highest component in the two groups identified, followed by p-cymene, terpinene, and carvacrol.Previous research produced identical results 33 .In the current study, storage conditions affected some chemical compounds' ratios of p-cymene, carvacrol, and thymol.Moreover, the highest carvacrol content was produced from 2.5 kGy of radiation at shade.Additionally, savory showed a significant increase in thymol in refrigerator condition.The fact that the amount of thymol was higher in the refrigerator than in the shade suggests that by adjusting plant storage conditions, one may keep a compound constant, reduce it, or increase it.The amount of p-cymene grew, and the amount of carvacrol decreased as plant storage time increased, which can be a crucial management point in creating the potent chemical.In other words, the quality of the essential oil (carvacrol) was diminished by using gamma rays for a longer time.The main essential oil constituents have a negative correlation, meaning their levels decrease as one increases.Therefore, duration and storage requirements, as well as corrective actions, will play a crucial role.The lack, or small effect, of irradiation on thyme aroma compounds agrees with Pereira et al. 34 .
According to our findings, gamma radiation significantly affected phenolic compounds and antioxidant activity.In addition, other compounds react and change besides essential oil as a volatile substance that evaporates through transpiration and humidity.Phenol is one of the antioxidant chemicals, and in this experiment, the stress condition was thermal stress.Phenol production increases under stress conditions, but as stress intensity increases, another pathway is activated, and phenol production decreases.According to Abbaszadeh et al. 35 , the proportion of essential oils and some physiological compounds rises under mild stress.As stress severity increases, new pathways become available, and the number of new compounds in the plant rises.A drop in phenol concentration at high radiation illustrates the same problem.Gamma rays have been shown to disrupt hormones, gas production, water absorption, and leaf enzyme activity.These actions modify plant metabolism, photosynthesis, cell structure, and phenolic chemical accumulation.In contrast, chloroplasts are significantly more sensitive to gamma rays than other cellular organelles, and the rise in pigment is very noticeable in samples exposed to radiation 17 .Due to the high concentration of phenolic and flavonoid components in the low-rate irradiation treatment, the highest antioxidant activity was seen.
In this regard, Kosalec et al. 36 found no appreciable differences in the amount of phenols and essential components of rosemary, artichoke spring grass, and basil by using the various gamma-ray intensities.Ebadi 23 stated that radiation had no discernible impact on the proportion of essential oils and their constituents, such as limonene and citral.In addition, the amount of antioxidants and total phenol was higher in the control samples.Gamma radiation at doses of 10 kGy on thyme 30 , 2 kGy on Stevia 37 , and 10 kGy on Thymus algeriensis 38 had a significant impact on essential oil alterations.Our findings agree with the study reporting that radiation less than 10 kGy does not alter carbohydrates 39.As a general statemen, essential oils with high concentrations of p-cymene and carvacrol are generally created using gamma-ray treatment with an intensity of 2.5 kGy in the relevant industries.Using a plant that has just been harvested may be a better choice if the thymol concentration is high.Changes in essential oil compositions (carvacrol and thymol para-cymene) were detected during storage, confirming that storage in medicinal plants is critical and that future research could be addressed.The decrease in thymol in the shade compared to the refrigerator indicates that preservation, increasing or decreasing EOs might be achieved by choosing plant storage conditions.Despite widespread publicity regarding the effectiveness of gamma rays in various plants, prospective studies may provide the most accurate information regarding the cost of transmission and the disadvantages of gamma rays on a large scale in the savory.Vol:.( 1234567890 https://doi.org/10.1038/s41598-024-57989-w

Figure 1 .
Figure 1.The effect of gamma ray intensity on the percentage of essential oil of S. mutica: means within each column followed by the same letter are not different according to the LSD test.Treatment: (0, 2.5.7.5 and 10 kGy of gamma radiation).

Figure 2 .
Figure 2. The effect of gamma radiation and storage conditions on the percentage of essential oil of S. mutica: means within each column followed by the same letter are not different according to the LSD test.Treatment: (0, 2.5.7.5 and 10 kGy of γ radiation); sh shade, Ref refrigerator.

Figure 3 .
Figure 3.The effect of gamma ray and storage time on the percentage of essential oil of S. mutica: means within each column followed by the same letter are not different according to the LSD test.Treatment: (0, 2.5.7.5 and 10 kGy of γ radiation); ST storing time: (0. 120, 240 h).
The authors declare that there is no conflict of interest regarding this article.

Table 2 .
The effect of gamma rays × storing conditions × storing time on the components of essential oil of S. mutica (units of all compounds are percentage).Means within each column followed by the same letter are not different according to the LSD test.

Table 3 .
The effect of gamma rays × storing conditions × storing time on the biochemical traits of S. mutica.Means within each column followed by the same letter are not different according to the LSD test.